Project Summary Osteoporosis is common and devastating, as 20% of adults with a hip fracture die within 1 year and another 50% never walk independently again. Medications to promote bone formation might be a superior approach to treating osteoporosis. Recombinant parathyroid hormone (PTH) and anti-sclerostin antibody increase bone formation by targeting PTH receptor (PTH1R) and Wnt signaling pathways, respectively. However, these medications require injections and can only be used for up to two years. In mice, combined treatment with PTH and anti-sclerostin antibody is more effective than either medication alone. Understanding the mechanisms by which combined PTH1R and Wnt signaling increase bone mass could lead to novel treatments to decrease fracture risk. We will test our central hypothesis that PTH1R is required for Wnt to fully stimulate bone formation. Mechanistically, we propose that PTH1R is required to stabilize the Wnt effector b-catenin in osteoprogenitors, which in turn maximally stimulates expression of the osteoblast gene program. We will use two approaches to activate Wnt signaling in mice lacking PTH1R in bone: pharmacologically with a novel water soluble Wnt surrogate, and genetically by knocking out the Wnt inhibitor sclerostin. We propose to use two innovative methods to overcome current barriers to understanding how PTH and Wnt signaling interact in bone. First, we will use mass cytometry (CyTOF) to analyze expression of >40 parameters, allowing us to distinguish mesenchymal stem cells, osteoprogenitors and osteoblasts, and to simultaneously examine the effects of PTH1R and Wnt signaling in these populations. Second, we will use single-cell RNA-sequencing to evaluate PTH1R and Wnt signaling in osteoprogenitors. We have preliminary data that in the absence of PTH1R signaling in osteoprogenitors, increased Wnt signaling fails to increase bone. In Specific Aim 1 we will determine whether intact PTH1R signaling is required for Wnt-dependent bone formation by pharmacological and genetic activation of Wnt signaling in 1 month-old control (PTH1ROsxWT) and PTH1ROsxKO mice. We will assess bone formation by histology, quantitative histomorphometry, and micro-computed tomography (CT). In Specific Aim 2 we will determine whether PTH1R is required for Wnt signaling to increase osteoprogenitor numbers by performing mass cytometry on bone cells of mice from Aim 1, using antibody panels to distinguish mesenchymal stem cells, osteoprogenitors and osteoblasts. We will simultaneously evaluate PTH1R and Wnt signaling in each population. We will use single-cell RNA-sequencing (scRNA-seq) to determine whether PTH1R is required for Wnt signaling to increase osteoblast gene programs in osteoprogenitors. In Aim 3 we will validate our findings in adult PTH1ROsxWT and PTH1ROsxKO mice treated with Wnt surrogate ligand, using CT, histomorphometry, mass cytometry and scRNA-seq. Successful completion of these aims will provide more detailed understanding of the mechanisms by which PTH1R and Wnt cooperate to increase bone formation.